Microparticles containing physiologically active peptide, method for preparing the same, and pharmaceutical composition comprising the same

ABSTRACT

Disclosed are microparticles containing physiologically active peptides, a method for preparing the same, and a pharmaceutical composition comprising the same.

TECHNICAL FIELD

The present invention relates to microparticles containingphysiologically active peptides, a method for preparing the same, and apharmaceutical composition comprising the same. More specifically, thepresent invention relates to microparticles which comprise an ioniccomplex of physiologically active peptide and water-soluble polymer, anda biodegradable water-insoluble polymer, and thus can reduce initialburst of peptide drugs and control the release rate, by which thetherapeutic effects of drugs can be improved; a method for preparing thesame; and a pharmaceutical composition comprising the same for peptidedrugs.

BACKGROUND ART

Conventional formulations for injection such as infusion solutions,suspensions and emulsions are rapidly removed from the body after theyare intramuscularly or subcutaneously administered. For this reason,frequent administration by injection is required for treatment ofchronic diseases. In an attempt to solve this problem,microencapsulation—a process in which a drug is encapsulated intomicrospheres—has been suggested. Microspheres are produced in a size ofunit of several μm to several tens of μm, and thus can be injectedintramuscularly or subcutaneously. Accordingly, they can control thedrug release rate and thus can adjust the drug delivery period.Therefore, a therapeutically effective drug concentration can bemaintained for a long period of time by a single administration.

Generally, after oral administration, most peptide drugs lose theiractive structures or are broken down by enzymatic degradation underacidic environments, and are poorly absorbed in the gastric orintestinal mucosa. For this reason, peptide drugs are administered byinjection. Peptide drugs should be injected continuously and repeatedlybecause of their short half-life and low bioavailability in vivo. It isalso frequently required to administer peptide drugs for a long periodof several months. Accordingly, a great deal of research on sustainedand controlled release formulations using biodegradable polymers isactively underway.

aliphatic polyesters have been developed and their bioavailabilty hasbeen approved by the U.S. Food and Drug Administration (U.S. FDA), andthey are currently used as polymeric carriers for peptide drugs.Aliphatic polyesters are widely utilized in applications includingcarriers for drug delivery, surgical sutures and the like.

Recently, physiologically active peptides have been developed as noveldrugs and thus a variety of approaches to continuously release the drugsby encapsulating these drugs in polymer carriers has been conducted.However, such formulations where peptide drugs are encapsulated inmicrospheres comprising an aliphatic polyester, have disadvantagesincluding initial burst (excessive release) effect of drugs, difficultyin maintaining the drug release rate constantly for a predeterminedperiod of time, and incomplete release, i.e., the release of less than100% of the encapsulated drug. The initial burst of drug is due to thefact that peptide drugs adsorbed on the surface and pores ofmicrospheres are rapidly diffused and released at an initial stage.Accordingly, there is a demand for a method for preparingsustained-release microspheres containing peptide, by which the initialburst of drugs is avoided and 100% of the encapsulated drug is releasedat a zero-order rate during the release period of time. It is alsorequired that the method is simple, the encapsulation ratio of the drugis high, the encapsulated drug is highly stable and the method iseconomically efficient.

It has been known that the methods for preparing drug-containingmicrospheres include phase separation, melt-extrusion, followed bycryopulverization, double emulsion evaporation (W/O/W, water/oil/water),single emulsion evaporation (O/W, oil/water), spray drying and the like.

The phase separation disclosed in U.S. Pat. No. 4,673,595 is a methodfor preparing microparticles by dissolving a polymer in methylenechloride. This method uses methylene chloride in combination withsilicone oil, ethyl alcohol, etc., and thus has a disadvantage of thecomplicated overall process to remove all of the used organic solvents.

U.S. Pat. No. 5,134,122 discloses the melt extrusion followed bycryopulverization. This method is free from the risk of residual toxicsolvents since no toxic solvent is used in the preparation process.However, peptides may be denatured due to the heat generated during thegrinding procedures to obtain PLGA microparticles and peptidemicroparticles. It is also difficult to control the size of the obtainedmicroparticles to a level suitable for easy injection.

The emulsion evaporation (W/O/W) is generally used. U.S. Pat. No.5,271,945 discloses a method in which an aqueous solution containing apeptide is dispersed in an organic solvent containing a biodegradablepolymer to form a primary emulsion (W/O) and the primary emulsion isthen dispersed in an aqueous phase containing an emulsifying agent.However, when microparticles are prepared by such emulsion evaporation,the microparticles may exhibit different biological response levels dueto the wide range of particle size distribution and thus it is difficultto predict pharmaceutical effects. Furthermore, it is difficult todevelop clinically useful carriers due to the existence of the particlesmuch bigger than the average size.

In addition, since most bio-peptide drugs are water-soluble, saidmethods have disadvantages in that a considerable amount of drugsescapes in the aqueous phase during the dispersing process and thus theencapsulation ratio becomes low.

DISCLOSURE OF INVENTION Technical Problem

Therefore, the present invention has been made in view of the aboveproblems, and it is an object of the present invention to providepolymeric microparticles containing physiologically active peptides anda biodegradable, water-insoluble polymer; and a method for convenientlypreparing the same. The microparticles of the present invention have auniform size, efficiently encapsulate physiologically active peptidestherein, reduce the initial burst of drug and exhibit the sustainedrelease for a predetermined period. In addition, the present method forpreparing the microparticles does not use an organic solventexcessively, and the residual minor amount of the organic solvent can beremoved efficiently, and thus any toxicity caused by organic solventscan be prevented. Also, the preparation can be conducted in a simplemanner.

Solution to Problem

In accordance with one aspect of the present invention, the above andother objects can be accomplished by the provision of a microparticlecomprising an ionic complex of physiologically active peptide andwater-soluble polymer, and a biodegradable, water-insoluble polymer.

In accordance with another aspect of the present invention, provided isa method for preparing physiologically active peptide-containing polymercomplex comprising: 1) mixing a physiologically active peptide with anionic water-soluble polymer in an aqueous medium to form an ioniccomplex of the physiologically active peptide and the water-solublepolymer; and 2) drying the ionic complex of the physiologically activepeptide and the water-soluble polymer obtained in step 1).

In accordance with another aspect of the present invention, provided isa method for preparing physiologically active peptide-containingmicroparticle comprising: a) homogeneously mixing an ionic complex of aphysiologically active peptide and a water-soluble polymer with abiodegradable, water-insoluble polymer in a non-aqueous solvent; and b)removing the non-aqueous solvent from the resulting solution obtained instep a) to obtain microparticle.

In accordance with yet another aspect of the present invention, providedis a pharmaceutical composition comprising the microparticle containingphysiologically active peptide according to the present invention, and apharmaceutically acceptable carrier.

Advantageous Effects of Invention

The microparticle containing physiologically active peptide according tothe present invention exhibits high encapsulation efficiency of peptidedrug, is of no initial burst of the peptide drug and can continuouslyrelease the drug for a long period of time. In addition, since themicroparticles are prepared in a relatively uniform size, the deviationbetween production batches decreases and thus the microparticles can beprepared with a uniform quality.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a scanning electronic microscope (SEM) image showing themicroparticles prepared in Example 9 of the present invention.

FIG. 2 is a scanning electronic microscope (SEM) image showing themicroparticles prepared in Comparative Example 3 of the presentinvention.

FIG. 3 is a scanning electronic microscope (SEM) image showing themicroparticles prepared in Comparative Example 4 of the presentinvention.

FIG. 4 is a graph showing the cumulative release amount of goserelinover time in Test Example 3 (in vitro long-period release test) of thepresent invention.

MODE FOR THE INVENTION

Hereinafter, the present invention will be described in detail.

Physiologically Active Peptide

As the physiologically active peptide contained in microparticles of thepresent invention, any water-soluble physiologically active peptideknown as a drug and a pharmaceutically acceptable salt thereof may beused without particular limitation. Examples of the physiologicallyactive peptide include, but are not limited to, LHRH analogs such asLHRH agonists or LHRH antagonists, somatostatin and analogs thereof,glucagon-like peptides (GLP), parathyroid hormone and analogs thereof,insulin-like growth factors, epidermal growth factors, platelet-derivedgrowth factors, fibroblast growth factors, transforming growth factors,growth hormone releasing factors, amylin analogs, peptide YY (PYY),protein synthesis-stimulating peptides, gastrin inhibitory peptides,vasoactive intestinal peptides and pharmaceutically acceptable saltsthereof.

Examples of the LHRH agonists include goserelin, leurporeline,triptorelin, buserelin, nafarelin, fertirelin, deslorelin, gonandorelin,alarelin, antide and the like, and examples of the LHRH antagonistsinclude cetrorelix, argitide, orntide and the like.

Examples of the glucagon-like peptides (GLP) include GLP-1, exenatide,liraglutide, taspoglutide, albiglutide, lixisenatide and the like.

Examples of somatostatin and analogs thereof include somatostatin,octreotide, lanreotide, vapreotide and the like.

Examples of vasopressin and analogs thereof include lypressin, oxytocin,argipressin, desmopressin and the like.

Other examples include calcitonin, elcatonin, corticotropin releasingfactors, brain natriuretic peptides, thymosin, thymopentin,corticotropin, beta-amyloid, angiotensin, atosiban, bivalirudin,cetrorelix, enfuvirtide, nesiritide, eptifibatide, secretin,teriparatide, terlipressin, tetracosactide, pramlintide and the like.

In the microparticles of the present invention, the physiologicallyactive peptide is preferably present in an amount of 1.0 to 10% byweight, more preferably, 2.0 to 5.0% by weight, with respect to thetotal weight of the microparticles. When the peptide is present in anamount less than 1.0% by weight with respect to the total weight of themicroparticles, the amount of microparticles to be administered to apatient increases excessively and thus the administration may beproblematic or impossible. When the peptide is present in an amountgreater than 10% by weight, the inhibition of initial burst may bedifficult.

<Ionic Water-Soluble Polymer>

In the present invention, an ionic water-soluble polymer is used forformation of an ionic complex with the aforesaid physiologically activepeptide. The ionic water-soluble polymer used in the present inventionis a water-soluble polymer which is ionized in an aqueous solution andthen takes positive or negative charge. It may form a complex havingionic bond with anion or cation of charged amino acid in thephysiologically active peptide.

The ionic water-soluble polymer is not particularly limited, andexamples thereof include, for example, polylactic acid having at leastone terminal carboxyl group or a derivative thereof. According to anembodiment of the present invention, the polylactic acid having at leastone terminal carboxyl group or a derivative thereof is one or moreselected from the group consisting of polylactic acid, polylactide,polyglycolide, polymandelic acid, polycaprolactone, polyanhydride andcopolymers thereof.

The remaining terminal group(s) other than the terminal carboxylgroup(s) of the polylactic acid or a derivative thereof is one or moreterminal groups selected from the group consisting of hydroxy, acetoxy,benzoyloxy, decanoyloxy, palmitoyloxy, methyl and ethyl. Since thepolylactic acid or a derivative thereof has anionic terminal carboxylgroup(s), it may form an ionic complex with the cationic amine group ofamino acid of the physiologically active peptide.

The number average molecular weight of the polylactic acid or aderivative thereof is preferably 500 to 5,000 daltons, more preferably1,000 to 3,000 daltons, and most preferably 1,000 to 2,000 daltons. Whenthe molecular weight is lower than 500 daltons, water-insolublity of theionic complex with the physiologically active peptide may beinsufficient. When the molecular weight is higher than 5,000 daltons,the polylactic acid or a derivative thereof is not dissolved in waterand thus it may be difficult to form the ionic complex. Accordingly, theabove range is preferred.

In the present invention, the polylactic acid having at least oneterminal carboxyl group or a derivative thereof is preferably one ormore selected from the group consisting of compounds represented byFormulae 1 to 6 below:

RO—CHZ-[A]_(n)-[B]_(m)—COOM  [Formula 1]

wherein A is —COO—CHZ—; B is —COO—CHY—, —COO—CH₂CH₂CH₂CH₂CH₂— or—COO—CH₂CH₂OCH₂; R is hydrogen atom, or acetyl group, benzoyl group,decanoyl group, palmitoyl group, methyl group or ethyl group; each of Zand Y independently is hydrogen atom, methyl group or phenyl group; M isH, Na, K, or Li; n is an integer of 1 to 30; and m is an integer of 0 to20,

RO—CHZ—[COO—CHX]_(p)—[COO—CHY′]_(q)—COO—CHZ—COOM  [Formula 2]

wherein X is methyl group; Y′ is hydrogen atom or phenyl group; p is aninteger of 0 to 25 and q is an integer of 0 to 25 provided that p+q isan integer of 5 to 25; R is hydrogen atom, or acetyl group, benzoylgroup, decanoyl group, palmitoyl group, methyl group or ethyl group; Mis H, Na, K, or Li; and Z is hydrogen atom, methyl group or phenylgroup,

RO—PAD-COO—W-M′  [Formula 3]

wherein W-M′ is

PAD is selected from the group consisting of D,L-polylactic acid,D-polylactic acid, polymandelic acid, copolymer of D,L-lactic acid andglycolic acid, copolymer of D,L-lactic acid and mandelic acid, copolymerof D,L-lactic acid and caprolactone, and copolymer of D,L-lactic acidand 1,4-dioxan-2-one; R is hydrogen atom, acetyl group, benzoyl group,decanoyl group, palmitoyl group, methyl group or ethyl group; and M isindependently H, Na, K, or Li,

S—O—PAD-COO-Q  [Formula 4]

wherein S is

L is —NR₁— or -0- in which R₁ is hydrogen atom or C₁₋₁₀ alkyl; Q is CH₃,CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃, or CH₂C₆H₅; a is an integer of 0 to 4;b is an integer of 1 to 10; M is H, Na, K, or Li; PAD is one or moreselected from the group consisting of D,L-polylactic acid, D-polylacticacid, polymandelic acid, copolymer of D,L-lactic acid and glycolic acid,copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lacticacid and caprolactone, and copolymer of D,L-lactic acid and1,4-dioxan-2-one,

wherein R′ is —PAD-O—C(O)—CH₂CH₂—C(O)—OM, in which PAD is selected fromthe group consisting of D,L-polylactic acid, D-polylactic acid,polymandelic acid, copolymer of D,L-lactic acid and glycolic acid,copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lacticacid and caprolactone, and copolymer of D,L-lactic acid and1,4-dioxan-2-one, and M is H, Na, K, or Li; and a is an integer of 1 to4, for example, 3-arm PLA-COONa provided a=1; 4-arm PLA-COONa provideda=2; 5-arm PLA-COONa provided a=3, and 6-arm PLA-COONa provided a=4,

YO—[—C(O)—(CHX)_(a)—O—]_(m)—C(O)—R—C(O)—[—O—(CHX′)_(b)—C(O)—]_(n)—OZ  [Formula6]

wherein each of X and X′ is independently hydrogen, alkyl (for example,alkyl having 1 to 10 carbon atoms such as methyl)or aryl (for example,aryl having 6 to 20 carbon atoms such as phenyl); each of Y and Z isindependently H, Na, K, or Li; each of m and n is independently aninteger of 0 to 95 provided that 5<m+n<100; each of a and b is eachindependently an integer of 1 to 6; and R is substituted orunsubstituted (CH₂)_(k)- in which k is an integer of 0 to 10, divalentalkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbonatoms, or a combination thereof.

In the present invention, the used amount of the ionic water-solublepolymer (for example, polylactic acid having at least one terminalcarboxyl group or a derivative thereof) is specifically from 0.1 to 10times of moles, more specifically 0.5 to 5 times of moles, and even morespecifically 1 to 2 times of moles, with respect to the used amount ofthe physiologically active peptide. When the used amount of the ionicwater-soluble polymer is less than 1 time of moles with respect to theused amount of the physiologically active peptide, there may be apeptide incapable of forming the ionic complex. When the used amount ofthe ionic water-soluble polymer is greater than 10 times of moles withrespect to the used amount of the physiologically active peptide, theprepared microparticles may be readily dissolved and released during asubsequent washing process and thus porosity of particlesdisadvantageously increases and, as a result, the release rate maydecrease. In addition, the ionic polymer present in the microparticlemay be decomposed to generate a considerable amount of acid that makesthe microenvironment in the microparticle acidic, which may thenincrease the degradation rate of other polymers present in themicroparticle and accordingly the peptide may be exposed to unstableenvironment.

<Ionic Complex of the Physiologically Active Peptide and theWater-Soluble Polymer>

In the present invention, the physiologically active peptide and theionic water-soluble polymer as described above form a complex throughionic bonding. For example, biodegradable polymeric polylactic acidhaving at least one terminal carboxyl group or a derivative thereof isreadily ionized in water and takes negative charge, and thus forms anionic bond with positively charged amino acid that constitutes thephysiologically active peptide. When the microparticles are prepared byusing the ionic complex prepared as such, peptide loss may be reduced,the encapsulation ratio of peptide can be improved and the initial burstcan be reduced. The complexes are present in the present microparticleas a distributed form therein.

Thus, in accordance with another aspect of the present invention,provided is a method for preparing physiologically activepeptide-containing polymer complex comprising: 1) mixing aphysiologically active peptide with an ionic water-soluble polymer in anaqueous medium to form an ionic complex of the physiologically activepeptide and the water-soluble polymer; and 2) drying the ionic complexof the physiologically active peptide and the water-soluble polymerobtained in step 1).

In the preparation method of the physiologically activepeptide-containing polymer complex, the physiologically active peptideand ionic water-soluble polymer used may be the same as those describedabove.

In step 1), the physiologically active peptide and the ionicwater-soluble polymer may be independently dissolved in separate aqueousmedia such as water to prepare respective aqueous solutions thereof andthese aqueous solutions may then be mixed. Alternatively, thephysiologically active peptide and ionic water-soluble polymer aresimultaneously added to an aqueous medium and mixed to prepare thecomplex. When the aqueous solution of the physiologically active peptideis separately prepared and used, pH of the aqueous solution ispreferably 4.0 to 10.0, more preferably pH is 4.5 to 7.5, and even morepreferably pH is 5.5 to 6.5.

In one embodiment of the present invention, when an alkali metal salt ofpolylactic acid having at least one terminal carboxyl group or aderivative thereof is used as the ionic water-soluble polymer, thealkali metal salt has a compound form in which the terminal carboxylgroup of the polylactic acid or a derivative thereof is ionically bondedto an alkali metal. Concretely, such a compound form may be obtained byneutralizing the polylactic acid having a terminal carboxyl group or aderivative thereof with sodium, potassium, lithium or the like. In thiscase, the alkali metal may be provided from sodium carbonate, sodiumhydrogen carbonate, potassium hydrogen carbonate, potassium carbonate orthe like.

The mixing ratio of the physiologically active peptide:the ionicwater-soluble polymer (for example, polylactic acid having at least oneterminal carboxyl group or a derivative thereof) is specifically 1:0.5to 10, more specifically 1:0.5 to 5, and even more specifically 1:1 to 2as a molar ratio. Problems which may occur when the mixing ratio isbeyond this range are described above.

In step 2), the drying may be carried out by a method such asfreeze-drying, vacuum drying or the like.

<Biodegradable, Water-Insoluble Polymer>

The biodegradable, water-insoluble polymer contained in themicroparticle of the present invention is a conventional polymer usedfor preparation of microparticles and a biodegradable aliphaticpolyester, which is insoluble in water but is hydrolyzed in vivo, isgenerally used. Such a biodegradable, water-insoluble polymer serves asa matrix containing the ionic complex of the physiologically activepeptide and water-soluble polymer as described above.

Specific examples of the biodegradable, water-insoluble polymers usefulfor the present invention include polylactide (PLA), polyglycolide(PGA), poly(lactide-co-glycolide) (PLGA) which is a copolymer thereof,polyorthoester, polycaprolactone, polydioxanone, polyalkylcarbonate,polyanhydride and copolymers thereof. Polylactide, polyglycolide andpoly(lactide-co-glycolide) which is a copolymer thereof are preferred.The biodegradable, water-insoluble polymers (in particular, polyesterssuch as PLA, PGA and PLGA) are biocompatible and stable substances sincethey are hydrolyzed in vivo and are converted into nontoxic lactic acidor glycolic acid. Their biodegradation rate can also be controlled toone to two weeks at minimum and one to two years at maximum, dependingon the molecular weight of polymers, ratio of monomers andhydrophilicity. Thus they are suitable for use in the present invention.One embodiment of the present invention uses poly(lactide-co-glycolide)in which the content of lactide is from 50% by weight to 85% by weightand the content of glycolide is from 15% by weight to 50% by weight.

The weight average molecular weight of the biodegradable,water-insoluble polymer is preferably 2,000 to 100,000 daltons, morepreferably 10,000 to 60,000 daltons, and even more preferably 25,000 to50,000 daltons. Intrinsic viscosity thereof is preferably 0.1 to 0.9dl/g, and more preferably 0.2 to 0.6 dl/g. When the intrinsic viscosityis too low, the polymer is degraded too fast and the physiologicallyactive peptide may not be continuously released for the desired period.When the intrinsic viscosity is too high, the polymer degradation is tooslow and thus the amount of released physiologically active peptide maybe too small, and accordingly the pharmaceutical effect may not beexerted.

<Physiologically Active Peptide-Containing Microparticles>

The physiologically active peptide-containing microparticle according tothe present invention contains the ionic complex of physiologicallyactive peptide and water-soluble polymer and the biodegradablewater-insoluble polymer.

The content of the ionic complex in the microparticle of the presentinvention is, with respect to the total weight of the microparticle(i.e., 100% by weight of the sum of the ionic complex and thebiodegradable water-insoluble polymer), specifically 4% by weight to 40%by weight and more specifically 8% by weight to 20% by weight, and thecontent of the biodegradable water-insoluble polymer is specifically 60%by weight to 96% by weight and more specifically 80% by weight to 92% byweight.

The content of physiologically active peptide encapsulated in themicroparticle is preferably 3 to 10% by weight, with respect to thetotal weight of microparticle, and encapsulation efficiency thereof ispreferably 60 to 99% by weight, more preferably 70 to 99% by weight, andeven more preferably 85 to 99% by weight, based on the amount of peptideused. The above encapsulation efficiency is equivalent to or higher thana level which is commonly obtained when a water-soluble active peptideis encapsulated in a biodegradable, water-insoluble polymer by aconventional microsphere preparation method. Furthermore, the presentinvention can resolve the conventional disadvantage when microparticlesare prepared using active peptide alone, including the increased initialburst of drug due to the increased porosity and the failure inprolonging a continuous release period to the desired level due to theincreased degradation rate of polymer.

The microparticle of the present invention may optionally contain asurfactant in order to uniformly disperse the ionic complex in themicroparticle. As the surfactant, those commonly used in the art may beused without particular limitation. Specifically, examples of usefulsurfactants include polymer surfactants such as poloxamers, polyvinylalcohol and polyoxyethylene sorbitan fatty acid esters, natural polymerssuch as gelatin and alkali salts of higher fatty acid. Such a surfactantmay be present, for example, in an amount of 0.001 to 1% by weight withrespect to 100 parts by weight of the microparticle.

The physiologically active peptide-containing microparticle according tothe present invention may be prepared by mixing the ionic complex ofphysiologically active peptide and water-soluble polymer with thebiodegradable, water-insoluble polymer in a non-aqueous solvent,followed by molding to micro-size and solvent removal.

Accordingly, in accordance with another aspect of the present invention,a method for preparing physiologically active peptide-containingmicroparticle comprising: a) homogeneously mixing an ionic complex of aphysiologically active peptide and a water-soluble polymer with abiodegradable, water-insoluble polymer in a non-aqueous solvent; and b)removing the non-aqueous solvent from the resulting solution obtained instep a) to obtain microparticle.

The non-aqueous solvent that can be used for the preparation method ofthe microparticle according to the present invention is not particularlylimited. Examples thereof include organic solvents such as methylenechloride, ethyl acetate, chloroform, acetone, N-methyl-2-pyrrolidone,N,N-dimethylformamide, tetrahydrofuran, dimethyl sulfoxide,hexafluoroisopropanol and mixtures thereof.

The amount of the biodegradable, water-insoluble polymer dissolved inthe non-aqueous solvent is, for example, 5% by weight to 50% by weightand more specifically 10% by weight to 30% by weight. When theconcentration is too low, an excessive amount of organic solvent is usedand removal of the organic solvent is thus difficult. On the other hand,when the concentration of the biodegradable, water-insoluble polymer istoo high, handling is difficult during the process and microwells maynot be readily filled with the solution during subsequentmicrofabrication.

In step a), sonication may be performed to obtain a homogeneousdispersion (or solution), if necessary.

In one embodiment, the removal of the solvent to obtain microparticlesin step b) can be carried out by a known microfabrication method(Acharya et. Al., J. Controlled Release, Vol. 141, Issue 3, Pages 314 to319, Korean Patent Laid-open Publication No. 10-2010-7009002).

According to the microfabrication, step b) comprises:

b-1) filling a plurality of microwells placed in a water-solublemicrotemplate with the solution obtained in step (a);

b-2) removing the non-aqueous solvent from the solution filled in themicrowells to solidify the biodegradable, water-insoluble polymer; and

b-3) collecting microparticles from the microtemplate.

The water-soluble microtemplate may be prepared by a known method. Forexample, an aqueous solution of water-soluble polymer such as gelatin,polyvinyl alcohol, agarose, poly(N-isopropyl acrylamide) and alginate isprepared (concentration may be about 1 to 80% by weight, varyingaccording to the type of polymer), and the prepared aqueous hydrophilicpolymer solution is applied to a silicone wafer provided withmicrostructures (that is, microwells) and solidified to give amicrotemplate provided with a plurality of microwells. The microwells inmicrotemplate prepared as such are three-dimensional structures whichhave a diameter of about 1 to about 200 μm and a height of about 1 toabout 200 μm. More specifically, the diameter may be about 20 to 100 μmand the height may be about 20 to 100 μm. Even more specifically, thediameter may be about 30 to 50 μm and the height may be about 30 to 50μm.

In case of a gelatin aqueous solution, an aqueous solution containing 20to 40% by weight of gelatin may be used, and in case of a polyvinylalcohol solution, 5 to 10% by weight of an aqueous solution or asolution of a mixed solvent of ethanol/water may be used.

In step b-1), the resulting solution (or dispersion) obtained in step a)is applied to the microtemplate prepared as above (for example, arectangle with a width of 4 cm and a length of 6 cm) to fill themicrowells of templates with the solution.

In step b-2), the non-aqueous solvent is removed from the solution tosolidify the biodegradable, water-insoluble polymer. The microtemplatefilled with the solution may be kept at room temperature to allow thenon-aqueous solvent to volatilize. Alternatively, the microtemplatefilled with the solution may be kept under reduced pressure to removethe non-aqueous solvent. Solidification of the solution may be carriedout at a room temperature or at a lower temperature.

In step b-3), the microtemplate having microwells filled with thesolidified substance is added to an aqueous medium (for example, water)to dissolve the water-soluble microtemplate and thereby collect themicroparticles.

If such a microfabrication is used, microparticles with a uniform sizecan be obtained and the possibility of remaining excessive organicsolvent is eliminated. In addition, the necessity of using excessivewater to collect the microparticles is eliminated and thus it ispossible to prevent a large amount of waste water from being generated.

In another embodiment, the removal of the solvent to obtainmicroparticles in step b) may be carried out by a known emulsionevaporation method.

According to the emulsion evaporation, step b) comprises: b-i) addingthe resulting solution obtained in step a) dropwise to an aqueoussolution of the water-soluble polymer in the presence of a surfactantwith stirring to remove the non-aqueous solvent and obtainmicroparticle.

As the aqueous solution of the water-soluble polymer, for example, anaqueous solution of water-soluble polymer such as gelatin, polyvinylalcohol, agarose, poly(N-isopropyl acrylamide) or alginate may be used(concentration may be about 0.1 to 10% by weight, varying according tothe type of polymer), and an aqueous solution of 0.1 to 5% by weight ofpolyvinyl alcohol is preferred.

The surfactant may be contained in the resulting solution obtained instep a), or in the aqueous solution of water-soluble polymer.

In such emulsion evaporation, when a polymer in an organic solvent isdispersed in an aqueous phase, the organic solvent is removed byextraction or evaporation and thus the polymer solubility decreases andthe polymer is solidified. As a result, microparticles are formed.

The method for preparing microparticles of the present invention mayfurther comprise a step of washing the obtained microparticles afterstep b). This subsequent washing step is carried out by using water orthe like. As a result of the subsequent washing, any impurities presentin the obtained microparticles and excessive peptides present on thesurface thereof can be removed.

In addition, the method may further comprise a step of drying themicroparticles by freeze-drying, vacuum drying or the like, after thewashing. Through such drying, possibly remaining moisture and organicsolvent can be removed further.

<Pharmaceutical Composition of Peptide Drug>

The microparticle of the present invention as explained above cancontinuously release the physiologically active peptide for apredetermined period, for example, preferably for at least several daysto several weeks or several months after administration. Themicroparticle of the present invention may be dispersed, delivered orapplied to the target site of the subject through a delivery route suchas injection and/or subcutaneous, intramuscular, intraabdominal ordermal implant, and intramucosal administration. For example, themicroparticle of the present invention may be administered in the formof a homogeneous suspension in a dispersion media such as an injectionsolution.

Accordingly, in accordance with another aspect of the present invention,provided is a pharmaceutical composition comprising the microparticlecontaining physiologically active peptide according to the presentinvention, and a pharmaceutically acceptable carrier.

For the pharmaceutical composition of the present invention, apharmaceutically acceptable carrier suitably selected from commonlyknown pharmaceutically acceptable carriers may be used, depending on theintended formulation. The pharmaceutically acceptable carrier maycomprise distilled water for injection, a thickner, an isotonic agent ora surfactant, and may contain a buffer if needed. For example, thepharmaceutically acceptable carrier is prepared by adding 3 to 5% ofcarboxymethylcellulose sodium, 0.9% (w/v) of sodium chloride, 0.1% to0.5% of polysorbate 20 and the like to distilled water for injection.The viscosity of the prepared carrier may be 20 to 100 cP at roomtemperature.

As explained above, for the physiologically active peptide contained inthe pharmaceutical composition of the present invention, anywater-soluble physiologically active peptide known as a drug and apharmaceutically acceptable salt thereof may be used without particularlimitation, and specific examples thereof are described above.Accordingly, the type of diseases that can be effectively treated and/orprevented by the pharmaceutical composition of the present inventiondepends on the physiologically active peptide used. For example, whenthe pharmaceutical composition contains an LHRH agonist among LHRHanalogs as the physiologically active peptide, the presentpharmaceutical composition suppresses secretion of sexual hormones suchas testosterone and estrogen, and thus can exhibit therapeutic and/orpreventive effects on prostate cancer, breast cancer, endometriosis andthe like which progress on the basis of hormone reactivity.

Hereinafter, the present invention will be described in more detail withreference to the following Examples. However, the examples are providedto illustrate the present invention only and should not be construed aslimiting the protection scope of the present invention.

EXAMPLES 1 to 11 Preparation of Microparticles According toMicrofabrication Method

(1) Preparation of Dispersion or Solution of Ionic Complex of GoserelinAcetate and Polylactic Acid or a Derivative Thereof, andPoly(Lactide-Co-Glycolide) (PLGA)

According to Table 1, goserelin acetate was dissolved in distilled waterat a concentration of 10 mg/mL and sodium polylactate (number averagemolecular weight of 1,500 Da) was dissolved in distilled water atconcentrations of 20, 30 and 40 mg/mL. The sodium polylactate wasprepared by a known method disclosed in Korean Patent Application No.2002-63955. 3 ml of the respective aqueous solutions were mixed witheach other such that the total volume was adjusted to 6 ml. The mixturewas freeze-dried for 24 hours. As a biodegradable water-insolublepolymer, poly(lactide-co-glycolide)(PLGA) 7525DLG 4E (manufactured byLakeshore Biomaterials) was added to a glass vial in an amount set forthin the following Table 1, and methylene chloride was added thereto,followed by dissolving 7525DLG 4E such that its concentration wasadjusted to 20% by weight. The freeze-dried product was added thereto,followed by mixing to completely dissolve or homogeneously disperse it.

TABLE 1 Goserelin Sodium Initial amount acetate polylactate 7525 DLG ofgoserelin Examples (mg) (1,500 Da) (mg) 4E (mg) added (%) Ex. 1 30 601400 2.0 Ex. 2 30 60 650 4.1 Ex. 3 30 60 410 6.0 Ex. 4 30 60 210 10.0Ex. 5 30 90 1400 2.0 Ex. 6 30 90 650 3.9 Ex. 7 30 90 380 6.0 Ex. 8 30 90180 10.0 Ex. 9 30 120 1400 1.9 Ex. 10 30 120 650 3.8 Ex. 11 30 120 3506.0

(2) Production of Water-Soluble Microtemplate

900 g of tepid distilled water was added to a 1 L vessel and 100 g ofpolyvinyl alcohol was slowly added thereto, while stirring. The solutionwas stirred with maintaining 40 to 50° C. to completely dissolve thepolyvinyl alcohol and thereby prepare an aqueous solution of 10%polyvinyl alcohol. 100 g of the aqueous solution of polyvinyl alcoholwas mixed with 100 g of ethanol and the mixture was allowed to be keptat 40 to 50° C. for 2 to 3 hours to prepare a 5% homogeneous PVAsolution.

4 ml of the 5% PVA solution prepared above was added to a silicon wafer(diameter: 7 cm) having three-dimensional microstructures (microwells).After leveling the surface of the wafer, drying was conducted in an ovenat 60° C. for 30 minutes. The dried PVA was separated from the siliconwafer to produce a microtemplate. The microtemplate had a disk shapewith a diameter of 7 cm and the microwells had a diameter of 50 μm and aheight of 50 μm.

(3) Application of Peptide-Containing Solution to Microtemplate

The microtemplate was fixed on a glass plate, 150 μl of the solution ofgoserelin acetate-containing ionic complex and biodegradable,water-insoluble polymer prepared in step (1) was applied to themicrotemplate repeatedly 5 to 10 times in a manner of straight line.

(4) Removal of Solvent and Collection of Microparticles

The microtemplate obtained in step (3) was kept at room temperature for4 to 5 hours to allow methylene chloride to volatilize. Then, themicrotemplate was added to 50 ml of tepid distilled water (35 to 45° C.)and shaken for 30 minutes to dissolve polyvinyl alcohol whichconstituted the water-soluble template, and then microparticles wereseparated according to the particle size by using 100 μm and 30 μmsieves. The microparticles having a particle size between 30 μm and 100μm were collected, placed in a centrifuge tube and washed with tepiddistilled water 2 to 3 times. The microparticles were centrifuged andfreeze-dried for 24 hours.

EXAMPLE 12 Preparation of Microparticles by Emulsion Evaporation Method

According to Table 2, goserelin acetate was dissolved in distilled waterat a concentration of 10 mg/mL, sodium polylactate (number averagemolecular weight: 1,500 Da) was dissolved in distilled water at aconcentration of 30 mg/mL. The respective aqueous solutions were mixedwith each other at a weight ratio of 1:1 such that the total volume wasadjusted to 6 ml. Precipitate was formed and freeze-dried for 24 hours.As a biodegradable water-insoluble polymer, poly(lactide-co-glycolide)(PLGA) 7525DLG 4E (manufactured by Lakeshore Biomaterials) was added toa glass vial in an amount set forth in the following Table 2, andmethylene chloride was added thereto, followed by dissolution such thatthe concentration of 7525DLG 4E was adjusted to 20% by weight. Thegoserelin acetate-containing freeze-dried product and 5.0 mg of asurfactant (Tween 80) were added thereto, and dissolved or dispersed byvigorous stirring to obtain a homogeneous liquid. The homogeneous liquidwas added dropwise to 500 mL of a 0.5% aqueous polyvinyl alcoholsolution at room temperature and, at the same time, vigorous stirringwas carried out by using a mixer for 20 minutes. The stirring speed wasdecreased, the reaction temperature was elevated to 40° C. and theorganic solvent added was removed for one hour. The resulting productwas centrifuged to collect microparticles, and the microparticles werewashed with 200 mL of tepid distilled water and centrifuged again toobtain final microparticles.

TABLE 2 Goserelin PLA-Na 7525 DLG Methylene Surfactant acetate (mg)(1,500 Da) (mg) 4E (mg) chloride (mg) (mg) 30 90 1,400 5,000 5

COMPARATIVE EXAMPLES 1 to 3 Preparation of Microparticles UsingGoserelin Acetate and PLGA (Microfabrication)

(1) Preparation of Dispersion or Solution of Goserelin Acetate and PLGA

According to Table 3, 30 mg of goserelin acetate, and 1400, 710 and 330mg of poly(lactide-co-glycolide)(PLGA) 7525DLG 4E (manufactured byLakeshore Biomaterials) as a biodegradable polymer, were added to glassvials, and methylene chloride was added thereto, followed by dissolutionsuch that the concentration of 7525DLG 4E was adjusted to 20% by weight.

(2) Application of Peptide-Containing Solution to Microtemplate andCollection of Microparticles

The same microtemplate as those used for Example 1 to 11 were fixed on aglass plate and 150 μl of a solution of goserelin acetate and abiodegradable polymer prepared in step (1) was applied to themicrotemplate in a manner of straight line. The application was repeated5 to 10 times and kept at room temperature for 4 to 5 hours to allowmethylene chloride to volatilize. Then, microparticles were collected inthe same manner as in Example 1 to 11.

TABLE 3 Goserelin 7525 DLG Initial amount of Comp. Ex. acetate (mg) 4E(mg) goserelin added (%) Comp. Ex. 1 30 1400 2.1 Comp. Ex. 2 30 710 4.1Comp. Ex. 3 30 330 8.3

COMPARATIVE EXAMPLE 4 Preparation Of Microparticles Using GoserelinAcetate and PLGA (Emulsion Evaporation)

Goserelin acetate was dissolved in tepid distilled water at aconcentration of 30 mg/0.3 mL to obtain an aqueous phase. 1,250 mg ofpoly(lactide-co-glycolide)(PLGA) 7525DLG 4E (manufactured by LakeshoreBiomaterials) as a biodegradable polymer, 5 mg of Tween 80 as asurfactant and 5 ml of methylene chloride were mixed with together anddissolved under vigorous stirring to obtain a clear solution. Thesolution was added to the aqueous phase in which goserelin acetate wasdissolved, followed by vigorous stirring to prepare an emulsion. Theemulsion was added dropwise to 500 mL of a 0.5% aqueous polyvinylalcohol solution at room temperature and, at the same time, vigorousstirring was carried out for 20 minutes by using a mixer. The stirringspeed was decreased, the reaction temperature was elevated to 40° C. andthe organic solvent added was removed for one hour. The resultingproduct was centrifuged to collect microparticles, the microparticleswere washed with 200 mL of tepid distilled water and centrifuged againto obtain final microparticles.

TEST EXAMPLE 1 Shape of Microparticles

In order to observe the surface of the prepared microparticles, about 10mg of microparticles of Example 9, and Comparative Examples 3 and 4 werefixed to an aluminum stub, coated with platinum under vacuum of 0.1 tonat high voltage (10 Kv) for 3 minutes and mounted in a SEM, and thesurface of the microparticles was observed using an image analysisprogram. The SEM images of microparticles are shown in FIGS. 1 to 3,respectively. It could be confirmed that the microparticles of Example 9had more uniform and less porous surfaces, as compared with those ofComparative Example 3. In addition, it could be confirmed thatmicroparticles of Comparative Example 4 had a considerably wide particlesize distribution, as compared with those of Example 9.

TEST EXAMPLE 2 Encapsulation Ratio of Goserelin in Microparticles

2 ml of methylene chloride was added to about 10 mg of microparticles ofExamples and Comparative Examples, followed by complete dissolution. 8ml of a 0.1M acetate buffer (pH 4.0) was added to the solution, shakenfor 10 minutes to transport goserelin to the aqueous solution layer andcentrifuged to collect the aqueous solution layer. The aqueous solutionlayer was filtered through a 0.45 μm syringe filter, the content ofgoserelin was measured by HPLC and the results are shown in Table 4. Thecolumn used herein was a CAPCELL PAK C18 (inner diameter: 2.0 mm,length: 15 cm), UV detection was performed at a wavelength of 230 nm,injected amount was 20 μl, and the flow rate of mobile phase was 0.2mL/min. The mobile phases used were (a) water containing 0.1% TFA and(b) acetonitrile containing 0.1% TFA, and the solvent ratio was changedsuch that the solvent (b) became from 0% to 65% for 30 minutes.

TABLE 4 Amount of Drug Initial amount goserelin encapsu- Goserelin ofgoserelin encapsu- lation Examples acetate:PLA-Na added (%) lated (%)ratio (%) Ex. 1 1:2 2.0 0.9 45 Ex. 2 1:2 4.1 1.6 39 Ex. 3 1:2 6.0 2.1 35Ex. 4 1:2 10.0 4.8 48 Ex. 5 1:3 2.0 1.6 80 Ex. 6 1:3 3.9 3.3 85 Ex. 71:3 6.0 3.0 50 Ex. 8 1:3 10.0 4.1 41 Ex. 9 1:4 1.9 1.6 84 Ex. 10 1:4 3.82.6 68 Ex. 11 1:4 6.0 3.2 53 Ex. 12 1:3 2.0 1.7 85 Comp. Ex. 1 1 2.1 0.524 Comp. Ex. 2 1 4.1 0.8 20 Comp. Ex. 3 1 8.3 1.0 12 Comp. Ex. 4 1 2.31.0 33

As can be seen from Table 4 above, Comparative Examples 1 to 4 exhibitedlow encapsulation ratios, as compared with Examples of the presentinvention, and in Comparative Examples 1 to 3, the amount of goserelinencapsulated in microparticles with respect to the total weight ofmicroparticles could not be increased to 1% or more, although theinitially amount of goserelin acetate added increased.

TEST EXAMPLE 3 In Vitro Long-Period Release Test of Microparticles

50 mg of microparticles prepared in Examples 1, 2 and 9 and ComparativeExample 3 were added to a test tube, 50 mL of a pH 7.4 phosphate bufferwas added thereto, stored in a stirrer set at 37° C., the supernatantwas collected after 6 hours, and 1, 3, 5, 7, 14, 21 and 28 days and theamount of released goserelin was measured by HPLC. The results are shownin FIG. 4.

As can be seen from FIG. 4, microparticles of Examples according to thepresent invention exhibited decreased initial drug release amounts andsuperior long-term continuous release properties. Thus, it could beconfirmed that they were remarkably advantageous as sustainedformulations, as compared with Comparative Examples.

1. A microparticle comprising: an ionic complex of physiologicallyactive peptide and water-soluble polymer; and a biodegradable,water-insoluble polymer.
 2. The microparticle according to claim 1,wherein the physiologically active peptide is selected from LHRHagonists, somatostatin and analogs thereof, glucagon-like peptides(GLP), parathyroid hormone and analogs thereof, insulin-like growthfactors, epidermal growth factors, platelet-derived growth factors,fibroblast growth factors, transforming growth factors, growth hormonereleasing factors, amylin analogs, peptide YY (PYY), proteinsynthesis-stimulating peptides, gastrin inhibitory peptides, vasoactiveintestinal peptides, and pharmaceutically acceptable salts thereof. 3.The microparticle according to claim 1, wherein the water-solublepolymer is polylactic acid having at least one terminal carboxyl groupor a derivative thereof, and is one or more selected from the groupconsisting of polylactic acid, polylactide, polyglycolide, polymandelicacid, polycaprolactone, polyanhydride and copolymers thereof.
 4. Themicroparticle according to claim 1, wherein the water-soluble polymer isselected from the group consisting of compounds represented by Formulae1 to 6 below:RO—CHZ-[A]_(n)-[B]_(m)—COOM  [Formula 1] wherein A is —COO—CHZ—; B is—COO—CHY—, —COO—CH₂CH₂CH₂CH₂CH₂— or —COOCH₂CH₂OCH₂; R is hydrogen atom,or acetyl group, benzoyl group, decanoyl group, palmitoyl group, methylgroup or ethyl group; each of Z and Y independently is hydrogen atom,methyl group or phenyl group; M is H, Na, K, or Li; n is an integer of 1to 30; and m is an integer of 0 to 20,RO—CHZ—[COO—CHX]_(p)—[COO—CHY′]_(q)—COO—CHZ—COOM  [Formula 2] wherein Xis methyl group; Y′ is hydrogen atom or phenyl group; p is an integer of0 to 25 and q is an integer of 0 to 25 provided that p+q is an integerof 5 to 25; R is hydrogen atom, or acetyl group, benzoyl group, decanoylgroup, palmitoyl group, methyl group or ethyl group; M is H, Na, K, orLi; and Z is hydrogen atom, methyl group or phenyl group,RO—PAD-COO—W-M′  [Formula 3] wherein W-M′ is

PAD is selected from the group consisting of D,L-polylactic acid,D-polylactic acid, polymandelic acid, copolymer of D,L-lactic acid andglycolic acid, copolymer of D,L-lactic acid and mandelic acid, copolymerof D,L-lactic acid and caprolactone, and copolymer of D,L-lactic acidand 1,4-dioxan-2-one; R is hydrogen atom, acetyl group, benzoyl group,decanoyl group, palmitoyl group, methyl group or ethyl group; and M isindependently H, Na, K, or Li,S—O—PAD-COO-Q  [Formula 4] wherein S is L is

—NR₁— or -0- in which R₁ is hydrogen atom or C₁₋₁₀ alkyl; Q is CH₃,CH₂CH₃, CH₂CH₂CH₃, CH₂CH₂CH₂CH₃, or CH₂C₆H₅; a is an integer of 0 to 4;b is an integer of 1 to 10; M is H, Na, K, or Li; PAD is one or moreselected from the group consisting of D,L-polylactic acid, D-polylacticacid, polymandelic acid, copolymer of D,L-lactic acid and glycolic acid,copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lacticacid and caprolactone, and copolymer of D,L-lactic acid and1,4-dioxan-2-one,

wherein R′ is —PAD-O—C(O)—CH₂CH₂—C(O)—OM, in which PAD is selected fromthe group consisting of D,L-polylactic acid, D-polylactic acid,polymandelic acid, copolymer of D,L-lactic acid and glycolic acid,copolymer of D,L-lactic acid and mandelic acid, copolymer of D,L-lacticacid and caprolactone, and copolymer of D,L-lactic acid and1,4-dioxan-2-one, and M is H, Na, K, or Li; and a is an integer of 1 to4,YO—[—C(O)—(CHX)_(a)—O—]_(m)—C(O)—R—C(O)—[—(CHX′)_(b)—C(O)—]_(n)—OZ  [Formula6] wherein each of X and X′ is independently hydrogen, alkyl or aryl;each of Y and Z is independently H, Na, K, or Li; each of m and n isindependently an integer of 0 to 95 provided that 5<m+n<100; each of aand b is each independently an integer of 1 to 6; and R is substitutedor unsubstituted —(CH₂)_(k)— in which k is an integer of 0 to 10,divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to20 carbon atoms, or a combination thereof.
 5. The microparticleaccording to claim 1, wherein the biodegradable, water-insoluble polymeris one or more selected from the group consisting of polylactide,polyglycolide, poly(lactide-co-glycolide), polyorthoester,polycaprolactone, polydioxanone polyalkylcarbonate, polyanhydride andcopolymers thereof.
 6. The microparticle according to claim 1, whereinthe physiologically active peptide is present in an amount of 1.0 to 10%by weight with respect to the total weight of the microparticle.
 7. Themicroparticle according to claim 1, wherein the content of the ioniccomplex is 4% by weight to 40% by weight, based on the total weight ofthe microparticle.
 8. A method for preparing physiologically activepeptide-containing polymer complex comprising: 1) mixing aphysiologically active peptide with an ionic water-soluble polymer in anaqueous medium to form an ionic complex of the physiologically activepeptide and the water-soluble polymer; and 2) drying the ionic complexof the physiologically active peptide and the water-soluble polymerobtained in step 1).
 9. The method according to claim 8, wherein thephysiologically active peptide and the ionic water-soluble polymer ismixed with a mixing ratio of 1:1 to 10 as a molar ratio.
 10. A methodfor preparing physiologically active peptide-containing microparticlecomprising: a) homogeneously mixing an ionic complex of aphysiologically active peptide and a water-soluble polymer with abiodegradable, water-insoluble polymer in a non-aqueous solvent; and b)removing the non-aqueous solvent from the resulting solution obtained instep a) to obtain microparticle.
 11. The method according to claim 10,wherein the non-aqueous solvent is selected from the group consisting ofmethylene chloride, ethyl acetate, chloroform, acetone,N-methyl-2-pyrrolidone, N,N-dimethylformamide, tetrahydrofuran, dimethylsulfoxide, hexafluoroisopropanol and mixtures thereof.
 12. The methodaccording to claim 10, wherein step b) comprises: b-1) filling aplurality of microwells placed in a water-soluble microtemplate with thesolution obtained in step (a); b-2) removing the non-aqueous solventfrom the solution filled in the microwells to solidify thebiodegradable, water-insoluble polymer; and b-3) collectingmicroparticles from the microtemplate.
 13. The method according to claim12, wherein the water-soluble microtemplate is produced from one or morewater-soluble polymers selected from gelatin, polyvinyl alcohol,agarose, poly(N-isopropyl acrylamide), alginate and mixtures thereof.14. The method according to claim 12, wherein, in step b-3), thecollection of microparticles is carried out by dissolving themicrotemplate in an aqueous medium.
 15. The method according to claim10, wherein step b) comprises: b-i) adding the resulting solutionobtained in step a) dropwise to an aqueous solution of the water-solublepolymer in the presence of a surfactant with stirring to remove thenon-aqueous solvent and obtain microparticle.
 16. A pharmaceuticalcomposition comprising the microparticle containing physiologicallyactive peptide according to claim 1, and a pharmaceutically acceptablecarrier.